Method for improving transformation efficiency of soybean

A technique for transforming soybeans and plants, applied in biochemical equipment and methods, botany equipment and methods, angiosperms/flowering plants, etc., can solve the problems of high false positive plant rate and low transformation efficiency

Active Publication Date: 2017-08-29
BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to provide a method for improving soybean transformation efficiency, which can effectively overcome the technical defects of high false-positive plant rate and low transformat

Method used

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  • Method for improving transformation efficiency of soybean
  • Method for improving transformation efficiency of soybean
  • Method for improving transformation efficiency of soybean

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Experimental program
Comparison scheme
Effect test

no. 1 example

[0140] The first embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium

[0141] 1. Construct a recombinant cloning vector containing the target gene

[0142] The SULE nucleotide sequence was connected to the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector DBN01-T, and its construction process Such as figure 1 Shown (wherein, Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ start codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; SULE is the sulfonylurea Herbicide-like hydrolase gene nucleotide sequence (SEQ ID NO: 1); MCS is a multiple cloning site).

[0143]Then, the recombinant cloning vector DBN01-T was transformed into Escherichia ...

no. 2 example

[0150] The second embodiment, the acquisition of transgenic soybean plants

[0151] Disinfection of soybean seeds: Take completely dried mature soybean seeds (Zhonghuang 13) and place them in a petri dish, accounting for about 1 / 3 of the petri dish. Place it in a desiccator in a fume hood, and place a 250ml beaker in the desiccator with 120ml of sodium hypochlorite inside. Add 6ml of concentrated hydrochloric acid dropwise along the wall of the beaker, close the desiccator and seal the lid, and close the glass of the fume hood at the same time, so that the soybean seeds are exposed to the chlorine gas in the fume hood to sterilize for 3 hours; Take out and shake for 2-3 minutes. Repeat the above process once, and put the Petri dish containing the soybean seeds in a fume hood to sterilize overnight.

[0152] Soybean seed germination: inoculate 15 soybean seeds after disinfection on the soybean germination medium (B5 salt 3.1g / L, B5 vitamin, sucrose 20g / L, agar 8g / L, pH5.6) to...

no. 3 example

[0176] The third embodiment, using TaqMan to verify the soybean plant transferred to the SULE nucleotide sequence

[0177] Take about 100 mg of the leaves of the above 12 kinds of treated soybean plants transferred to the SULE nucleotide sequence as samples, extract the genomic DNA with Qiagen's DNeasy Plant Maxi Kit, and detect the copy of the SULE gene by the Taqman probe fluorescence quantitative PCR method number. At the same time, the wild-type soybean plants were used as a control, and the detection and analysis were carried out according to the above method. The experiment was repeated 3 times, and the average value was taken.

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Abstract

The invention relates to a method for improving the transformation efficiency of soybean. A method for selecting transformed plant cells comprises the following steps: transforming the plant cells by using a recombinant carrier of a gene containing a target gene and a gene for encoding sulfonylurea herbicide hydrolase; carrying out screening culture on the transformed plant cells by externally applying an ALS (Acetolactate Synthetase) inhibitor, and using the gene for encoding the sulfonylurea herbicide hydrolase as a selected marker; selecting the plant cells which are not killed and/or not inhibited. According to the method disclosed by the invention, a method of adding a selective agent into an enrichment culture medium and a differential culture medium by an external application mode (in particular to dropping) in the plant transforming process is put forward for the first time, and effective concentration screening range of the selective agent is optimized, and the proportion and the transformation efficiency of positive plants obtained by an offspring are remarkably improved; meanwhile, transgenic plants obtained by transformation using the sulfonylurea herbicide hydrolase as the selected marker have the advantages of high commercial value, good resistance and genetic stability.

Description

technical field [0001] The invention relates to a method for plant transformation, in particular to a method for improving soybean transformation efficiency by externally applying a screening agent. Background technique [0002] With the rapid increase of transgenic plant species and planting area, the biosafety of selectable marker genes in transgenic plants has become one of the problems that people generally pay attention to. Appropriate marker genes can provide a powerful judgment basis for obtaining true transformants, and play a crucial role in the process of plant genetic transformation. At present, marker genes widely used in plant genetic transformation include antibiotic resistance genes (such as NPTⅡ gene, HPT gene, etc.) and herbicide resistance genes (such as PAT gene, EPSPS gene, bar gene, etc.). Success is no longer useful, and even there is a potential threat to the ecological environment and food safety, so the development of biosafety marker genes is very ...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00
CPCC12N15/8205C12N15/821C12N15/8274C12N15/8275C12N15/8277C12N15/8278
Inventor 刘雁华王媛媛贾志伟宋庆芳
Owner BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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